Method and device for a accomodating samples on cryosubstrates

ABSTRACT

For sample picking on a cryosubstrate, on which multiple cryopreserved samples are each positioned at preselected sample positions, individual samples are selectively separated mechanically or thermally from the cryosubstrate and transferred to a target substrate.

BACKGROUND OF THE INVENTION

The invention concerns a method for sample picking on cryosubstrates,particularly a method for transferring samples in a cryopreserved orthawed condition from a cryosubstrate to a target substrate. Theinvention also concerns a device for implementing a method of this typeand a cryosubstrate which is functionally textured for sample taking.

The operation of cryobanks for preserving biological cell material isgenerally known in cell biology, molecular biology, and geneticengineering. In a cryobank, the cell material is kept available fordecades, with, for example, suspended cells being frozen in small-volumecontainers (volumes range from 0.1 ml to a few ml) filled with acryoliquid. In order to ensure the viability of the cell material afterthawing, numerous procedures have been developed which, for example,relate to the timing of the thawing, media additives, container shapes,and similar things. With conventional cryobanks, survival rates rangingfrom a few percent up to 90% are achieved during thawing. Although thisis already a relatively good result and cryobanks have found worldwidedistribution, the following disadvantages are connected with thecryopreservation procedures disseminated until now.

The position of individual cell material samples in the volume of thecryoliquid is unknown during both the freezing and the thawingprocedures. The material samples are not accessible in the preserved,deep frozen state. However, there is interest in, for example, removingsingle cells from cryopreserved material, measuring, or changing them.However, in order to be able to remove cells, the entire sample must bethawed. This requires costly recultivation of the cell material tocompensate for the thawing losses. Over the course of time, thecryopreserved material thus no longer contains only the originallypreserved cells, but a mixture of daughter cells of greatly varyinggenerations, which restricts the specificity and reproducibility of cellinvestigations. In order to subject all material samples in acryocontainer to the same cooling progression, extremely slow freezingprocedures must be provided, since the cooling proceeds from thecontainer walls and all samples in the cryovolume are to experienceapproximately the same temperature progression over time. Finally, thesuspension medium (cryoliquid) prevents or makes more difficultmeasurement and processing of single cells at low temperatures.

There is an interest in new cryopreservation technologies for overcomingthe disadvantages mentioned and for opening new fields for cellpreservation, particularly since researches in biotechnology, geneticengineering, and medicine are increasingly directed toward single cells,such as in hybridoma cell production in connection with tumor treatment,stem cell culture, and embryogenesis. The development of newcryopreservation technologies is based on the following knowledge andconsiderations.

From a physical and physiological viewpoint, a cell frozen at −196° C.is in a solid state. The metabolic processes have come to a completestandstill down to the molecular level. Cell changes only arise throughslow restructuring (e.g. through the growth of ice crystals attemperatures above −80° C.) and through damage due to cosmic radiation.The latter, however, has a rate of approximately 90% damage after 30,000years, which is non-critical for practical applications. In the deepfrozen state, cells should therefore able to be measured, treated,changed, sorted and otherwise manipulated in a mechanically robust waywithout time pressure and with the highest precision. However, thisassumes the ability to individually handle the cells in the cryomediumand the availability of tools for cell manipulation.

The physical and chemical procedures during the freezing or thawing ofbiological materials are, for example, described in the publication ofF. Franks “Biophysics and biochemistry of low temperature andfreezing”in “Effects of Low Temperatures on Biological Membranes”(EditorG. J. Morris et al., Academic Press, London, 1981) or P. Mazur in “Ann.N.Y. Acad. Sci.”, vol. 541, 1988, p. 514 et seq. The prevention of theformation of intracellular or extracellular ice crystals and excessivedehydration of the cells is decisive for freeze preservation over longperiods of time and thawing with the greatest possible survival rate. Inthis case, the following characteristics are to be taken intoconsideration from a physical viewpoint during freezing and thawing.Producing so-called vitrified water, in which any type of ice crystalformation is suppressed, through extremely high freezing speeds isknown. However, this cannot be used for careful and positionally definedfreezing of cell material, since the size of the biological cells ofinterest and heat conduction restricts the freezing speeds to valuesbelow a few ten thousands of degrees per second. Therefore, at themicroscopic scale and under physiological conditions, segregation, i.e.formation of eutectic phases, which also include domains of pure ice,can be observed. To minimize the segregation, cell-specific freezingprograms have provided the best results, particularly at the beginningof cooling (down to −30° C.), (see also S. P. Leibo et al. in“Cryobiol.”, vol. 8, 1971, p. 447 et seq). In this temperature interval,cooling rates of a few degrees per minute have been shown to be morefavorable than rapid temperature jumps. It is inferred from this thatthe cooling and thawing procedures should be performed with abiologically specific temperature profile over time.

As soon as temperatures at which ice formation begins have been reached,however, higher cooling rates are appropriate, since in this way themigratory growth of larger ice domains at the cost of smaller ones canbe prevented. At temperatures below the range of −80° C., no further icecrystal growth occurs, so that cell storage over long periods of time ispossible. The storage of the container with cell material which issuspended in a cryoliquid is typically performed in liquid nitrogen (at−196° C.). Since the sample container is closed, there is no directcontact with the liquid coolant phase. Comparable temperature sequencesare used for thawing the cell material.

Cooling procedures are also known from preparation for electronmicroscope recordings (see D. G. Robinson et al. in“Präparationsmethodik in der Elektronenmikroskopie”, Springer-Verlag,Berlin, 1985). In contrast to cryopreservation, which has the goal ofmaintaining the vitality of the cells, in electron microscopy, the leastpossible change in the molecular position of the cell components playsthe decisive role. Therefore, during this preparation, particularlyrapid freezing technologies are realized, which include, for example,shooting the sample into liquid or undercooled gases or spraying dropsinto an undercooled atmosphere and liquids. In this case, cooling ratesof more than 10,000 degrees per second are achieved, which, however, dueto the cell volume, the finite thermal conductivity, and the wettabilityof the material, represent a limiting value.

A general problem in cryopreservation is that not all types of cells canbe cryopreserved in the same way. In particular, larger objects (cellgroups or the like) or cells containing large numbers of vacuoles, whichparticularly occur in plant sample material, can be revitalized onlywith difficulty or not all. The development of new microinjection andcell handling technologies, as well as new cryoprotectives, is directedtoward these problems. A technology which is different from thepreservation in containers described above is based on the freezingand/or thawing of the cell material to be preserved in adhered form oncooled surfaces (see, for example, T. Ohno et al in “Cryotechnol.”, vol.5, 1991, p. 273 et seq).

Cryopreservation on cooled surfaces is more difficult to handle than thesuspension principle, but has been shown to have advantages in theinvestigation of the processes occurring during cryopreservation and inachieving higher survival rates during thawing. Cryopreservation onsubstrate surfaces allows the boundary conditions of the respectiveprocedure, such as surface temperature, thermal conduction, cell ordroplet size, etc., to be adjusted and detected more exactly and morevariably than in the suspension of a cryomedium. This is particularlyused in cryomicroscopy, with biological cells which are enclosed in thesolvent drops being misted or sprayed onto frozen surfaces (see H.Plattner et al in “Freeze-etching, Techniques and Application”, editorE. L. Benedetti et al, “Soc. Franc. Microsc. Electronique”, Paris 1973,p. 81 et seq, and PCT/US94/01156). A disadvantage of the initiallydeveloped cryopreservation on substrate surfaces is that the positionand arrangement of the cells cannot be controlled during misting orspraying and multiple drops and cell layers can even be deposited on topof one another.

An improvement of cryopreservation on substrate surfaces is described inEP 804 073. Biological cells surrounded by an enveloping solution areplaced using a microdroplet jetting device in a predetermined way onsubstrates the temperature of which can be adjusted. The microdropletjetting device, which can be driven like an inkjet printer, allows ahighly precise and reproducible positioning of individual materialsamples on the cryosubstrate. Texturing the cryosubstrate with recessesapplied in a matrix in order to allow specific procedures duringcryopreservation and/or during thawing of the substrate is also knownfrom EP 804 073. The recesses are thus particularly adapted for directeddeposition of the cells. To produce test chips, with which theinteraction of greatly differing cells in the thawed state is to beinvestigated, various cell types are deposited in or between therecesses. Furthermore, providing electrodes for implementing highfrequency electric fields at the recesses, under whose effect aninvestigation of the cells in the thawed state can be performed, isknown from EP 804 073.

Cryopreservation on cooled surfaces has had the disadvantage until nowthat, after the application onto the cryosubstrate, a sample-specifichandling of single cells was only possible in the deep frozen or thawedstate on the cryosubstrate. If processing in the thawed state wasintended, the entire substrate had to be heated. However, forimprovement of the investigation techniques and increased utilization ofcryopreserved sample stocks, it is important to make the individualmaterial samples accessible to specific handling.

SUMMARY OF THE INVENTION

The object of the invention is to provide an improved method for samplepicking on cryosubstrates which particularly allows selective taking ofpreselected samples or sample groups from a cryosubstrate. The object ofthe invention is also to provide devices for implementing a method ofthis type.

These objects are achieved by a method and/or devices with featuresaccording to the patent claims, and/or. Advantageous embodiments andapplications of the invention arise from the dependent claims.

According to the invention, predetermined, selective sample pickingoccurs on a cryosubstrate with multiple samples which are located onpredetermined sample positions through positionally specific mechanicalor thermal separation of the samples from the cryosubstrate and transferof the released samples to a target substrate. Sample picking is herebygenerally understood to mean any type of picking or taking of samples,if necessary with certain parts of the substrate.

Any device which is suitable as a carrier for samples frozen onto cooledsurfaces is referred to in this case as a cryosubstrate (or: carriersubstrate, substrate). It serves for sample preservation or storage. Thecryosubstrate includes a carrier material for arranging the samples inlinear or planar shapes with a functional surface texture described indetail below. According to a preferred embodiment, the carrier materialconsists of an inert material, such as plastic, ceramic, metal, orsemiconductor material, which can be structured with a mechanical orchemical processing means known per se. The cryosubstrate preferablyforms a rigid, planar, flat or curved body which is bonded in a wayknown per se with a temperature adjusting device. Alternatively, thecryosubstrate can, however, also be made of a flexible, film-likecarrier material, for example plastic.

The carrier material is preferably implemented integrally with thesurface texture (or: structure), but can also include a combination ofthe materials described in specific embodiments. This combination can,for example, be an electrically insulating base material with specificsurface coatings made of, for example, metal. For the realization of thepresent invention, a functional surface texture (or: structure) isgenerally understood to mean any type of geometrical or material changeof the cryosubstrate through which localized deposition regions arecreated, corresponding to the sample positions on the cryosubstrate,from which the respective sample or samples can be selectively removedwithout the entire cryosubstrate having to be heated. The sample pickingaccording to the invention therefore preferably occurs on cryosubstratesin the deep frozen operating state.

The method according to the invention can be implemented with any typeof sample desired which can be applied onto a cryosubstrate while deepfrozen (e.g. at the temperature of liquid nitrogen). The samplespreferably consist of biological material, such as biological cells orcell groups or cell components, if necessary each with an envelopingmedium. The invention can, however, also be used with syntheticmaterials, such as vesicles, or with combinations of biological andsynthetic materials.

The sample picking and transfer according to the invention occurs towarda target substrate, which refers to, in general, any type of device forfurther handling or manipulation of the sample. For example, storage,mechanical or chemical processing, or investigation of the sample occurson the target substrate. The target substrate can thus also be acryosubstrate of a further preservation system.

A positionally selective mechanical separation of samples from thecryosubstrate includes separation of predetermined deposition elementsfrom the substrate with the respective samples or sample groups. Theseparation occurs with a suitable tool, preferably while maintaining thecryopreserved state of the samples. However, a mechanical separation inthe thawed sample state can also be provided. A thermal separationoccurs according to a first embodiment of the invention through apositionally specific increase of the substrate temperature in such away that the appropriate sample is thawed and removed with a suitabletool (e.g. micropipette, picking needle), or that positionally specificdeposition elements with preserved samples are thermally separated fromthe substrate. For thermal separation, electrical resistance heating orradiation heating (laser, microwaves, or the like) is used on thedesired sample position. In an alternative form of thermal sampleseparation, freezing of the desired samples onto a textured tool, whichproduces stronger adhesion of the frozen samples than the adhesion to asample carrier, is provided.

A subject of the invention is also a sample picking or sample handlingsystem for picking and/or transferring samples from a cryosubstrate ontothe target substrate, with a system of this type particularly includinga functionally textured cryosubstrate, a separation device, and acontrol device. The separation device serves as a separation deviceand/or as the picker for the separated or released sample.

According to a particularly important aspect of the invention, acryosubstrate is provided with a functionally textured surface whichincludes multiple deposition elements (e.g. deposition plates,deposition films), which are each implemented for accommodating onematerial sample and for selective mechanical or thermal separation ofthe sample, if necessary with a part of the deposition element, from thecryosubstrate. The dimensioning of the deposition elements is selecteddepending on the application. A deposition element can havecharacteristic dimensions of a magnitude from 1 cm² to 1 mm² or evenless. The separation of entire sample groups from the cryosubstrate canalso be provided.

The invention has the advantage that for the first time the restrictionsof cryopreservation on temperature stabilized substrate surfaces areovercome and selective processing of individual samples is madepossible. In this way, the effectiveness of single cell cryopreservationis significantly increased. The design of a cryosubstrate according tothe invention is based on well controllable texturing methods that areknown per se. The cryosubstrate can be produced as a disposable productfrom economical material. A further advantage concerns the ability toautomate the overall system. Through the combination of sample pickingwith an image processing system, sample transfer from a cryosubstrate toone or more target substrates can be performed independently of theoperator and automatically.

BRIEF DESCRIPTION OF THE DRAWINGS

Further advantages and characteristics of the invention are shown in theembodiments described in the following with reference to the attacheddrawings.

FIG. 1 shows a schematic overview of a device according to the inventionfor sample picking on cryosubstrates,

FIG. 2 shows a cryosubstrate according to the invention withmechanically separable deposition elements,

FIG. 3 shows an enlarged perspective view of a deposition element shownin FIG. 2,

FIG. 4 shows an illustration of the transfer of individual samples froma cryosubstrate according to an embodiment of the method according tothe invention,

FIG. 5 shows a further cryosubstrate according to the invention withmechanically separable deposition elements,

FIG. 6 shows an enlarged perspective view of four deposition elements asshown in FIG. 5,

FIG. 7 shows a further cryosubstrate according to the invention which isimplemented for simultaneous separation of multiple samples,

FIG. 8 shows a further cryosubstrate according to the invention in theform of a flexible film,

FIG. 9 shows a further cryosubstrate according to the invention which isadapted for thermal sample separation,

FIG. 10 shows an enlarged illustration of a heating element as shown inFIG. 9,

FIG. 11 shows an illustration of a further embodiment of the methodaccording to the invention using the cryosubstrate shown in FIG. 9,

FIG. 12 shows a schematic sectional view of a further embodiment of acryosubstrate according to the invention with thermal separation ofdeposition elements,

FIGS. 13, 14 show further surface textures on cryosubstrates,

FIG. 15 shows a schematic sectional view of a cryosubstrate according tothe invention with individually movable deposition elements,

FIG. 16 shows surface textures for influencing the adhesiveness of thesamples on cryosubstrates, and

FIG. 17 shows an illustration of a further method for sample pickingaccording to the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention is described in the following with reference to thehandling of samples which, for example, include one or more biologicalcells with an enveloping solution droplet and which are cryopreserved atthe temperature of liquid nitrogen. The invention can be implemented ina corresponding way with the further sample materials described above.Restriction to a specific temperature range, or a specific temperatureregime during freezing, storage, and thawing of the samples is notindicated. Details of these procedures are known per se and can berealized by those skilled in the art depending on the application.

FIG. 1 is a schematic overview of a device according to the inventionfor sample picking on a cryosubstrate. The device specifically includesthe cryosubstrate 100 having multiple deposition elements 200, which areeach designed for cryofixed storage of a sample 300, a separation device400, an image recording device 500, and a control system 600. Thecryosubstrate 100, whose design forms are described in detail below, canhave its temperature controlled with a cooling and/or heating assembly101in a way known per se and, if necessary, can be moved with amechanism (not shown) in a storage device. The separation device 400 isdesigned for mechanical or thermal separation of samples from thecryosubstrate 100 and for transfer of the separated samples onto one ormore target substrates 130. A drive unit 401 is provided for moving theseparation device 400. Depending on the application, the separationdevice 400 can, however, also be manually operated. The provision of thedrive unit 401 is a facultative feature of the invention which, however,can particularly be implemented advantageously in automated samplepicking procedures on cryosubstrates. The image recording device 500 isarranged for recording an image of the surface of the cryosubstrate 100.In the control system 600, an image evaluator known per se is providedwith which the surface image recorded can be evaluated in regard to thepositions of the samples to be picked. The driving of the drive unit 401can occur depending on the sample positions determined. The control unit600 is further connected with the cooling and/or heating assembly 101,the cryosubstrate 100 (for thermal sample separation), a display 601,and possibly with the target substrates 130. In place of or in additionto the image recording device 500, an observation system, e.g. amicroscope, can be provided for visual observation of the substratesurface.

According to a preferred embodiment of the invention, at least oneseparation device, such as a pipette head or needle head of a pickingrobot, can travel over the cryosubstrate and be operated at the desiredsample positions. Multiple separation devices can also be positioned andactuated in a matrix like a pipette matrix with a picking robot.

A first embodiment of the cryosubstrate 100 according to the inventionis shown enlarged in a schematic perspective view in FIG. 2. Thecryosubstrate 100 includes a substrate body 110 with a surface textureformed by the deposition elements 200. The substrate body 110 forms arigid, flat body and is made of plastic (e.g. PMMA), ceramic (e.g.aluminum oxide and other sintered ceramics), metal (e.g. titanium,silver), or a semiconductor material (e.g. silicon). Ceramic andsemiconductor materials have the advantage of good thermalcharacteristics, with high thermal conductivity particularly beingsought for effective cooling.

The deposition elements 200 are positioned in a matrix in rows andcolumns. Depending on the application, altered geometries of the layout(e.g. circular, in groups, or the like) can be implemented. In theembodiment illustrated, the deposition elements 200 are formed bydeposition plates 210. Details of a deposition plate 210 are illustratedin FIG. 3.

The deposition plates have the shape of a stamp or mushroom and includea carrier 211, which has a smaller cross-section, rising out from thesubstrate body 110, which carries a deposition lamina 212 of largercross-section on the side away from the substrate body 210. Thedeposition lamina 212 has a centrally located recess 213 foraccommodating the sample 300, which in the example illustrated includesa frozen enveloping solution droplet 310 with a cell 320. The referencenumber 321 refers to the schematically illustrated cell nucleus of thecell 320.

The design and dimensions of the deposition plate 210 are selecteddepending on the application, particularly in consideration of thedesign of the separation device (see below). The carrier 211 isimplemented with a cross-section small enough that, during-theseparation of the sample 300 from the cryosubstrate according to theinvention, it forms a mechanical predetermined break point. In contrast,the deposition lamina 212 is implemented thicker and wider, so thatduring separation of the carrier 211, no damage to the deposition lamina212 occurs.

When a forked separation device (see FIG. 4) is used, the depositionlamina preferably has the rectangular shape illustrated. However, around shape can also be provided, particularly if a capillary separationdevice is used for sample picking.

The deposition elements 200 are preferably formed integrally with thesubstrate body 110 through a suitable structuring process. With acryosubstrate based on silicon, the procedure is, for example, asfollows. First, the substrate body 110 having a thickness ofapproximately 0.1 mm to 1.5 mm, e.g. based on a wafer material, isproduced and provided with an SiO₂ film (thickness approximately 0.1 μmto 5 μm). This SiO₂ film is selectively etched according to the desiredintervals of the deposition lamina 212 (see FIG. 2), so that the Simaterial of the substrate body 110 is exposed according to the row andcolumn shape between the deposition elements 200. Underetching of thecover layer occurs in these exposed regions, so that the stamp shapeillustrated is implemented.

The sample picking from cryosubstrate 100 is schematically illustratedin FIG. 4. The separation device 400 shown in a detail has a beam 410with a forked separation tool 411 on its end pointing toward thecryosubstrate 100. The separation device 400 can be moved manually orwith the drive unit 401 (see FIG. 1) in all three spatial directions inrelation to the cryosubstrate 100. The beam 410 is alignedperpendicularly or at a slant to the substrate surface. The separationtool 411 extends essentially parallel to the substrate surface andincludes two prong-like projections which are implemented for thepurpose of engaging under one deposition plate 210 at a time andseparating (breaking) it from the substrate body 110 when a specificpulling or shearing force is exercised. The separation force can, asillustrated, be generated by mechanical leverage or alternatively byexercising a vacuum on the respective deposition element, e.g. with amicropipette.

In detail, the sample picking occurs with the steps of moving theseparation device 400 to the desired substrate position (arrow A),breaking or separating of the deposition plate 210 (arrow B), transferof the samples picked (with the deposition element) to the targetsubstrate 130 (arrow C) and storage and/or further manipulation of thesample on the target substrate 130. A gap 113 results corresponding tothe sample removed in the cryosubstrate 100′.

In order that the sample 300 remains in the cryopreserved state duringtransfer, it can be provided that the separation device 400 itself iscooled and/or the transfer occurs with blowing of a cold stream ofnitrogen. In an altered design of the separation device 400 (not shown),it has a sleeve-shaped separation tool (e.g. the tip of a micropipette)which moves over the desired deposition plate 210 from above and breaksit off with a slight shearing movement.

An alteration of the picking of samples with parts of the cryosubstratedescribed in FIGS. 2 to 4 is given by the following design, not shown.The functionally structured surface of the cryosubstrate has plasticfilm pieces positioned in a line or matrix as deposition elements, whichare each glued flat like an adhesive strip onto the substrate body. Thepieces of film have a size selected depending on the application, likethat, for example, of the deposition plates 210 according to FIG. 2. Forsample picking, a suitable stripping tool with a cutting edge or bladeis used which engages under the edge of the desired film piece and pullsit off of the cryosubstrate with the sample. The film is glued onto thesubstrate body with a suitable adhesive. Affixing the film so it adhereswithout an adhesive is, however, also possible. Alternatively, theentire substrate body can also be covered with a flat film, from whichindividual pieces are cut out for selective sample picking, as isdescribed with reference to FIG. 8.

FIG. 5 illustrates a further embodiment of a cryosubstrate 100 accordingto the invention having a surface-textured substrate body 110, in whosesurface recesses 112 having a wedge-shaped cross-section are etchedand/or undercut in rows in such a way that unsupported depositiontongues 214 are formed as the deposition plates 210. Each depositiontongue is arranged to accommodate one or (as shown) more samples 300. Anenlarged illustration of the deposition tongues 214 is shown in FIG. 6.Each deposition tongue is textured with three recesses 213, in each ofwhich one cell sample 320 is located.

The deposition tongues 214 have a predetermined break point on theirends pointing toward the substrate body 110, at which the separationusing a suitable tool occurs. The separation device is again preferablyequipped in this case with a forked separation tool or also with agripping or clamping tool or a suction device for picking the depositiontongues 214.

The production of a cryosubstrate corresponding to the embodimentillustrated in FIGS. 5 and 6 using semiconductor material occurs throughanisotropic etching of the recesses 112 (undercutting of the depositiontongues 214).

The letters A-D in FIG. 6 indicate a possibility of marking for theindividual deposition elements of the cryosubstrate. The marking makesthe orientation of the operator during observation of the cryosubstratethrough a microscope and, possibly, also image evaluation in the controlsystem 600 (see FIG. 1) easier.

The picking in groups of one sample or multiple samples, respectively,from a cryosubstrate 100 is illustrated in FIG. 7 in a schematic sideview (upper part of the image) and a horizontal projection (lower partof the image). The cryosubstrate 100, e.g. in the form of a wafer,carries a texture on its surface in the shape of linear tapers 215,which divide the substrate surface into segments 216 arranged in rowsand columns. The tapers 215 form intended break points which allowselective separation of individual samples or sample groups arranged inrows or columns. The areas filled out in black schematically illustratedepressions 213 corresponding to the depressions 213 of the depositionlaminas 212 and/or the deposition tongues 214 described above. Eachdepression 213 is again intended to accommodate one sample in thecryopreserved state.

The implementation of the tapers 215 occurs, depending on the substratematerial, through a suitable texturing process, e.g. through etching,milling, or the like. The reference number 217 relates to aschematically drawn region which is used for cryopreservation on thecryosubstrate 100.

A further embodiment in which mechanical separation of a part of thesubstrate on which a sample is deposited is provided is illustrated inFIG. 8. The cryosubstrate 100 is formed by a substrate film 120. Thesurface texture (not shown) of the substrate film 120 includes acircular or frame-shaped separating line at each intended sampleposition, at which the substrate film 120 is preferably cut throughand/or a grid-shaped marking network is produced by printing samplepositions. In this design, for picking samples according to theinvention, it is provided that the substrate film 120 be cut around thedesired sample with the separation device 400 and the sample betransferred with the section of the substrate film to the targetsubstrate. The separation device 400 is a cutting device or, as is shownin the example, an optical means in the form of a laser beam 413 focusedon the substrate film 120. The laser beam allows cutting through thesubstrate along the cutting line 121, like a mechanical cutting device.The substrate part cut out is picked with a picker, e.g. a micropipettewith a vacuum applied to it, and transferred to the target substrate.

In the following, an embodiment of the sample picking according to theinvention is described with reference to the FIGS. 9 to 14, in which athermal separation of the desired samples (possibly with parts of thesubstrate) occurs. In this case, various designs are provided whichallow the samples to be picked in the deep frozen state or in the thawedstate.

In the embodiment shown in FIG. 9, the cryosubstrate 100 carriesdeposition elements 200 in the form of multiple heating elements 230positioned in rows and columns, which each have a heating region 231, aground connection 232 implemented jointly for all heating elements 230,and a control connection 233. The heating region 231, the groundconnection 232, and the control connection 233 are shown enlarged inFIG. 10. These components again form a functional texture on the surfaceof the cryosubstrate 100 in which sample deposition at specific samplepositions corresponding to the location of the heating region 231 andthermal sample separation upon electrical current flow through therespective heating element 230 is provided. The substrate material ismade of, for plastic or ceramic. The heating elements 230 can be formedfrom any suitable, preferably inert, conductive material (e.g.platinum). The ground connections 232 in rows are preferablyelectrically connected with one another via a ground line 234surrounding the entire cryosubstrate 100.

Each of the heating elements 230 again has characteristic dimensions inthe cm to mm range, but can also be implemented significantly smaller,down to the μm range. The respective heating region 231 is formed by anarrow, preferably meander-shaped, conductor strip, which heats up whencurrent flows between the control connection 233 and the groundconnection 232. The control connection 233 is implemented as a touchpadwhich can be subjected to a voltage for achieving the desire heatingcurrent through the heating region 211 applied to it by placing amovable electrode on it (see below).

The selective thermal sample separation is also illustrated in theschematic perspective view shown in FIG. 11. FIG. 11 again shows thesubstrate 100 with the substrate body 110, which carries the heatingelements 230 positioned in rows and columns. The ground connections 232are all connected with the negative pole of a heating current source421. The positive pole of the heating current source 421 is connectedwith a tracer electrode 422 of the separation device 420, otherwise onlyschematically shown with dashed lines, for positionally selectivethermal release of samples from the cryosubstrate 100. The tracerelectrode 422 can be moved together with the separation device 420 orseparate from it in relation to the cryosubstrate 100. The placement ofthe tracer electrode 422 on one control connection 234 of a heatingelement 230 at a time provides a current flow and therefore heating ofthe heating region 231, so that the sample (not shown) positioned on theheating region 231, thaws partially or completely from the substrate andcan be picked with the separation device 420. This device is preferablydesigned as a micropipette.

The tracer principle illustrated in FIG. 11 can be modified as follows.It can be provided that, in place of the single tracer tip 422, a groupof tracer tips for releasing a group of samples is used according to apreselected pattern. Furthermore, a plug principle can be realized inplace of the tracer principle. It is also possible to provide each rowof heating elements with a joint ground connection separated from theconnections of the other rows and to provide a joint control connectionseparated from the connections of the other columns for each heatingelement column. In this design, the sample release occurs in such a waythat the heating current source 421 is connected by a control devicewith a row-column pair whose intersection point exactly corresponds tothe position of the desired sample.

FIG. 12 shows an altered embodiment of the thermal sample pickingaccording to the invention, in which a part of the deposition element isalso separated from the cryosubstrate with each sample. Thecryosubstrate 100 includes the substrate body 110 and the depositionelements 200 as a surface texture in the form of electrically detachabledeposition plates 220. Each deposition plate 220 includes a carrier 221and a deposition lamina 222. The deposition lamina 222 is provided witha recess 223 for accommodating the sample 300, as in the embodimentsdescribed above. Each carrier 221 includes a separating element 224 anda connection element 225, which is separated by the separating element224 from the substrate body 110. The separating element 224 is made of,for example, electrically conductive components, which, upon heating dueto a current flowing through them, melt or decompose or at least allowseparation of the deposition lamina 222 from the substrate body 110.

The substrate body 110 is made completely or partially from metal and isprovided with an electrical connection 117, which is electricallyconnected on the substrate side with all of the separating elements 224.Each connection element 225 is provided with its own electrical controlconnection 226. If a connection pair 117, 226 of a specific depositionelement 220 now has a voltage applied to it, the current flow throughthe separating element 224 thus causes it to melt or weaken, so that thedeposition element 200 affected can be picked with a suitable tool (see,for example, FIG. 4) and transported to the target substrate. This isillustrated in the lower part of FIG. 12.

The separating element 224 preferably consists of a material whichchanges due to the current flow (e.g. dissolves), such as a non-noblemetal (aluminum or the like), a gel, etc., and has a thickness of lessthan 0.5 mm.

The advantage of the arrangement according to FIG. 12 is that, in spiteof the thermal sample picking, the sample 300 can remain in thecryopreserved state.

Alterations of a functional cryosubstrate with surface-integratedheating elements which are individually controllable according to thesample positions are illustrated in FIGS. 13 and 14 in a schematic topview. According to FIG. 13, the substrate body 110 of the cryosubstrate100 carries straight electrode strips 240 which alternately encloseregions of reduced electrical conductivity 241 and regions of elevatedelectrical conductivity 242. The electrode strips have a characteristicwidth which corresponds to the typical transverse dimension of thedeposition surface of the cryopreserved samples. The samples 300 arepositioned in the regions 241 of reduced electrical conductivity. Incontrast, the regions of elevated electrical conductivity 242 formtracer connections and/or supply points for a heating current.

With two movable tracer tips analogous to the tracer electrode 422 inFIG. 10 or with a suitable line circuit, it is provided for samplepicking according to the invention that two regions of elevatedelectrical conductivity 242 at a time have a heating voltage applied tothem. The current flow between the two driven regions provides heatingin the region of reduced electrical conductivity 241 between the drivenregions 242, so that the samples positioned there are released. At thesame time, multiple sample regions can also be included, as isillustrated by the arrows 243, 244, which show two electrical tracerelectrodes which are each connected with the connections of a heatingcurrent source. Thus, the strip design shown in FIG. 13 also allows therelease of sample groups positioned in rows. Sample picking then againoccurs with a suitable tool, such as a micropipette or a picking needle,on whose tip the sample adheres. FIG. 14 shows an alteration of theprinciple shown of deposition of the samples on substrate regions oflower electrical conductivity, which are electrically connected withneighboring regions of elevated electrical conductivity, in the exampleof a cryosubstrate 100 having multiple openings 115 in the substratebody 110. The positions of the openings correspond to the desired sampledeposition positions. The openings 115 have a smaller diameter than thecharacteristic cross-sectional dimensions of the samples to be deposited(e.g. for the deposition of biological cells smaller than 100 μm). Theopenings 115 are provided with a coating, on both sides of the substratebody 100, which forms a region of reduced electrical conductivity 246.The coatings on both substrate sides are electrically connected with oneanother. Otherwise, the substrate body 100 is provided. on both sides ofthe substrate with a coating which forms one or more regions of elevatedelectrical conductivity 247. The regions 247 allow driving of individualdeposition positions (247 a) or of sample groups (247 b). For thispurpose, the electrically conductive, preferably metallic coating forforming the regions 247 is cut through at the desired positions (e.g. at248) depending on the application (introduction of slot-shapedinterruptions or the like).

By applying an electrical heating voltage to the continuous coating ofhigher conductivity on the back of the substrate on one side and of thedesired region 247 corresponding to a preselected sample, the desiredheating current flows over the warming regions 246, which causes atleast partial thawing of the sample and therefore its release.

A further embodiment of a functionally textured cryosubstrate 100 forselective sample picking is shown enlarged in a schematic side view inFIG. 15. The substrate body 110 of the cryosubstrate has multipleopenings which are positioned, for example, in a matrix in rows andcolumns according to the desired sample deposition. The depositionelements 200 are formed in this embodiment by movable depositionplungers 250, which are each movably located in one of the openings.Each deposition plunger 250 includes a carrier rod 251 and a depositionlamina 252, which is possibly provided with a recess (corresponding tothe recess 213 shown in FIG. 2 or 3) or with a surface texture as shownin FIG. 16 (see below). The deposition laminas 252 are arranged foraccommodating the cryopreserved samples 300, which, in the exampleshown, again include an enveloping solution droplet 310 with abiological cell 320.

In the initial state and/or in the storage state of the cryosubstrate atlow temperatures, all deposition plungers 250 sit in the correspondingopenings 115 in such a way that the deposition laminas 252 rest on thesurface of the substrate body 110. The length of the rod element 251 isgreater than the thickness of the substrate body 110, so that the rodelements 251 project out of the lower side of the substrate in theinitial state.

For selective (sample-specific) sample picking or sample picking ingroups, single deposition plungers 250 or groups of deposition plungers250 are now mechanically lifted from the substrate surface from the rearside of the cryosubstrate 100. This state is illustrated in the lowerpart of FIG. 15 for four deposition plungers. The deposition plungers250 pushed forward can then be lifted with a suitable separation orgripping tool, such as that described above with reference to FIG. 4,and transferred to the target substrate. At the same time, thecryopreserved state of the samples can be maintained.

It can be advantageous for the various sample picking procedures on thecryosubstrates to anchor the samples with a higher or lower retentionforce on the cryosubstrate. For this purpose, the substrate is texturedat the sample deposition positions, so that the contact region betweenthe substrate and sample is enlarged or modified to achieve the desiredretention forces. Examples of these types of textures are shown in FIG.16.

According to structure a, a nanotextured or micro-textured roughening261 is provided on the surface of the substrate body 110. Thisroughening 261 is produced, for example, with a chemical treatment or alaser treatment of the substrate and serves for better adhesion of thesample 300. According to the detail b, an extremely smooth surface isprovided which, for example, is formed by a polished region 262. Insidethe polished and possibly partially hydrophobic region 262, the sample300 can be easily displace or separated from the substrate, even in thedeep frozen state. This makes changing the position or picking thesample on the cryosubstrate easier. According to detail c, the sample300 is deposited in a trough 263, which serves both for improvedanchoring on the substrate and for protection against, for example, thetools during separation of neighboring samples. The texture 260 of thesubstrate can also include a profiled opening 264 according to detail d.The opening 264 has a smaller diameter than the biological cell 320contained in the sample 300. Since, however, the enveloping solutiondroplet 310 can at least partially penetrate the opening 264 during thefreezing procedure, a particularly strong anchoring of the sample in thecryopreserved state results. The detail e illustrates further substrateprofiles in the form of cups or trenches, which serve to influence thedroplet shape during the freezing process and/or to solidly anchor thesample to the substrate.

FIG. 17 shows a further variant of sample picking according to theinvention on cryosubstrates, which particularly serves for producingsample patterns on the cryosubstrate. In the uppermost image of FIG. 17,a sample carrier 140 of any desired type is shown, which carriesmultiple samples 300 at normal temperature (liquid state of the samples300). Each sample 300 includes, for example, an enveloping solutiondroplet 310 and two cells 320. The sample pattern on the sample carrier140 is, for example, produced with a picking robot with the aid ofmicropipettes.

After completion of the pattern, a deep frozen cryosubstrate 100 isplaced on the samples 300 (central image in FIG. 17), so that thesamples 300 freeze. The cryosubstrate 100 has textures 260 forincreasing adhesion on the surface facing the samples, as was described,for example, in FIG. 16. The sample carrier 140, in contrast, has asmooth, preferably polished surface. During the freezing process, thesamples 300 therefore adhere more strongly to the cryosubstrate 100 thanto the sample carrier 140 and can therefore be lifted with thecryosubstrate 100 (lowermost image of FIG. 17).

The method illustrated in FIG. 17 has the advantage of a definedfreezing procedure (cryoprocedure) for all of the samples. In addition,the droplets have a planar surface after adhesion on the cryosubstrate,which is particularly advantageous for microscopic observation.

The embodiments described above of the mechanical and/or thermal samplepicking according to the invention through separation of single ormultiple samples (possibly with parts of the substrate) from thecryosubstrate can particularly, depending on the application, bemodified for specific cryosubstrate shapes (e.g. cylindrical surfaces)or for specific separation tool shapes. Furthermore, it can be providedthat the sample picking according to the invention is combined with ameasurement method in which the cryopreserved samples are examined onthe cryosubstrate in regard to specific properties and are thenautomatically removed from the cryosubstrate.

A cryosubstrate according to the invention can, depending on theapplication, be adapted in regard to its material, shape, size, andsurface design for specific measurement tasks. Thus, for NMRexaminations, for example, it can be provided that the cryosubstrate ismade of an inert material suitable for NMR examinations, and is tailoredin size and shape to the respective available NMR measurement devices.Furthermore, a marking of cryosubstrates or of their parts, e.g. in theform of barcodes, color codes, visually detectable patterns, orelectromagnetic markings (transponders) can be provided. Thisadvantageously allows automatic detection of preselected samples onspecific deposition elements and/or the detection of the specificpositions from which samples are to be picked.

According to a further embodiment of a cryosubstrate according to theinvention, deposition elements which are designed for positionallyspecific separation from the cryosubstrate can include a magneticmaterial. Magnetic deposition elements can easily be detected with amagnet at the end of an appropriate picking device and transferred tothe respective target substrate.

The features of the invention described in the preceding description,the drawings, and the claims can be of significance both individuallyand in any desired combination for the implementation of the inventionin its various embodiments.

What is claimed is:
 1. A method of sample picking on a cryosubstrate, onwhich multiple cryopreserved samples are each located at preselectedsample position, comprising the steps of selectively separating singlesamples mechanically or thermally from the cryosubstrate andtransferring the samples to a target substrate.
 2. A method according toclaim 1, wherein, for mechanical separation of the samples, depositionelements, on each of which a sample is located, are selectively removedwith a separation device from a substrate body of the cryosubstrate byexercising mechanical pulling or shear forces and each sample thuspicked is transferred together with the deposition element to the targetsubstrate.
 3. A method according to claim 2, wherein the removal of thedeposition elements includes breaking off of deposition plates, whichare connected with the substrate body via predetermined break points, orpulling off of deposition films.
 4. A method according to claim 2,wherein the removal of the deposition elements includes selectivedisplacement of deposition plungers and picking of the displaceddeposition plungers with a gripping device.
 5. A method according toclaim 2, wherein the removal of the deposition elements includesselective cutting out of deposition regions from a film substrate andpicking of the cut out regions with a gripping device.
 6. A methodaccording to claim 1, wherein, for thermal separation of the samples, atdeposition elements, on each of which a sample is located and which areformed by hearing elements, a selective, at least partial thawing of therespective sample with the heating element occurs.
 7. A method accordingto claim 6, wherein the heating elements each have a control connectionand the sample picking includes placement of a tracer electrode on thecontrol connection for selective heating of the appropriate sample andpicking of the sample with a separation device.
 8. A method according toclaim 1, wherein, for mechanical separation of the samples, depositionelements, on each of which a sample is located, are selectively removedwith a separation device from a substrate body of the cryosubstrate byexercising a thermal decomposition and the respective sample picked istransferred together with the deposition element to the targetsubstrate.
 9. A method according to claim 1, wherein a portion of thecryosubstrate in a region of the samples remaining on the cryosubstrateremains at a cryogenic temperature during the sample picking.
 10. Amethod according to claim 2, wherein sample groups are selectivelypicked.
 11. A method according to claim 6, wherein sample groups areselectively picked.
 12. A method according to claim 8, wherein samplegroups are selectively picked.
 13. A device for sample picking oncryosubstrates, which includes: a functionally surface-texturedcryosubstrate having multiple deposition elements for cryopreservedsamples, with the deposition elements being implemented for selectivemechanical or thermal separation of the samples from the cryosubstrates,and a separation device which is implemented for separating and pickingthe samples from the cryosubstrates.
 14. A device according to claim 13,wherein the deposition elements include deposition plates, which areeach connected via a predetermined break point with a substrate body ofthe cryosubstrate.
 15. A device according to claim 14, wherein theseparation device include,des a forked separation tool, a gripping tool,a clamping tool, or a suction device.
 16. A device according to claim13, wherein the deposition elements include deposition plungers, whichare displaceably positioned in a substrate body of the cryosubstrateperpendicular to its surface.
 17. A device according to claim 16,wherein the separation device includes a forked separation tool, agripping tool, a clamping tool, or a suction device.
 18. A deviceaccording to claim 13, wherein the deposition elements include heatingelements, which are implemented for at least partial thawing of therespective deposited sample.
 19. A device according to claim 18, whereinthe heating elements each have a heating region, which is connected witha ground connection and a control connection, with all groundconnections of the heating elements being electrically connected withone another and the control connections being selectively electricallyseparated from one another and able to have a heating voltage applied tothem for application to the heating elements.
 20. A device according toclaim 13, wherein the deposition elements include electrically removabledeposition plates, in each of which a separation element is provided,which, upon application of an electrical voltage, allows separation ofthe sample with the deposition plate from the substrate body throughmelting or decomposition of the separation element.
 21. A cryosubstratewhich forms a carrier for multiple samples located on a surface of thecryosubstrate in a frozen state, wherein the surface has multipledeposition elements for accommodating one sample each, with eachdeposition element being designed for selective separation of therespective sample from the cryosubstrate.
 22. A cryosubstrate accordingto claim 21, wherein the deposition elements include deposition plateswhich are connected via predetermined break points with a substrate bodyof the cryosubstrate.
 23. A cryosubstrate according to claim 21, whereinthe deposition elements include heating elements which are designed forat least partial thawing of the respective deposited sample.
 24. Acryosubstrate according to claim 21, wherein the deposition elementsinclude deposition plungers, which are as displaceable in a substratebody of the cryosubstrate perpendicular to a surface of thecryosubstrate.
 25. A cryosubstrate according to claim 21, wherein filmpieces are provided as the deposition elements, which can be pulled offof the substrate body in a cooled state of the cryosubstrate.
 26. Acryosubstrate according to claim 21, wherein the deposition elements onthe substrate surface include a surface modification for influencing thesample adhesion, said surface modification being a surface roughening, apolishing, a recess, an opening, or an anchoring trench.
 27. Acryosubstrate according to claim 21, wherein the deposition elements aremade at least partially from magnetic materials.
 28. A cryosubstrateaccording to claim 21, on whose surface optical or electromagneticmarkings are provided for identifying the cryosubstrate and/orindividual deposition elements on the cryosubstrate.